To evaluate the efficiency and safety of promising and novel gene therapy approaches, the comprehensive testing of gene-modified cells in validated preclinical in vivo models is crucial. The conservation of relevant cell surface markers'cross-reactivity of reagents, and ability to apply the same supportive treatments administered to patients along nonhuman primate models to closely mimic clinical parameters. Demonstrating the procedure with me will be Anai Perez, a research technician from Dr.Hans-Peter Kiem's laboratory.
Begin by adding 10 to 12 milliliter aliquots of bone marrow isolated from a healthy nonhuman primate donor into 50 milliliter conical tubes. Bring the volume of the tube up to 50 milliliters with hemolytic buffer, and incubate the cells for up to seven minutes at room temperature. Remove the cell debris by centrifugation, and aspirate all but the last two to five milliliters of the supernatant.
Resuspend the pellet in the residual supernatant volume by flicking the tube, and transfer the bone marrow suspensions into a new 50 milliliter tube using a 70 micrometer pore cell strainer to remove any blood clots. Add fresh hemolytic buffer to bring the volume up to 50 milliliters, and incubate the cells for another five minutes at room temperature before their collection by centrifugation. Aspirate all of the supernatant and resuspend the cells in 10 milliliters of max buffer.
Before filtering the cell suspension through another 70 micrometer pore strainer, rinse the tube with 40 milliliters of fresh buffer, and pool the wash with the cell suspension. After enriching for CD34 positive cells according to standard magnetic assisted cell sorting protocols, wash the bead isolated cells in up to 50 milliliters of fresh max buffer, and resuspend the pellet in HSPC medium. Then plate 10 to 20 milliliters of the cells into vented tissue cultured treated T75 flasks for their overnight culture in a 37 degrees celsius and 5%carbon-dioxide incubator.
To assess the pre and post enrichment purity of the bone marrow's cell samples create a flow cytometry protocol with the appropriate plots and population hierarchy showing all of the gates for all events scatter, CD34 positive, hematopoietic stem cells, multipotent an-erythro myeloid progenitors and lympho myeloid progenitors. Next, run an unstained, pre-enrichment, white blood cell sample to adjust the voltages for the forward and side scatter parameters, and all of the fluorescence channels followed by single stained compensation beads to adjust the compensation between adjacent channels. Then run all of the remaining unstained and stained samples with the adjusted and compensated protocol to document the CD34 enrichment efficiency.
When all of the compensations have been set configure the cell sorter for the sort purification of the CD34 subsets into colony forming cell assays, and load the stained CD34 enriched sample tube onto the cytometer. Then record 2000 to 3000 events, and fine adjust the sort gates to fit the signal strength and target populations. When the setup is complete acquire the cells adjusting the flow rate to 500 to 1000 cells per second, and sorting 800 to 1200 cells from the scatter, CD34 positive, hematopoietic stem cell, multipotent an-erythro myeloid progenitor and lympho myeloid progenitor gates into separate tubes containing 3.6 milliliters of colony forming cell medium per tube.
At the end of the sort vortex the collection tubes, and add one milliliter of cell suspension to three sterile 3.5 centimeter, non-tissue culture treated petri dishes set within individual 15 centimeter petri dishes per cell population for their 10-14 day incubation at 37 degrees celsius. The next morning use a 10 milliliter pipette to gently rinse the adherent cells from the bottom of each flask with sterile HBSS, and collect the harvested cells by centrifugation. Resuspend the cells in transduction medium at a 1 x 10 to the 6th cells per milliliter concentration.
And add one milliliter of cells for the mock control in one well of a recombinant fibronectin fragment coated 12 well plate for an overnight incubation in the cell culture incubator. Then add 10 milliliter aliquots of the remaining cells into recombinant fibronectin fragment coated T75 flasks for a 30 minute incubation in the cell culture incubator with the caps vented. During the incubation thaw virus conditioned medium samples and calculate the amount of virus to be added based on the titer and the number of infectious units of cells per milliliter.
Take the flask out of the incubator and add the appropriate volume of virus conditioned medium to each flask before returning the cells to 37 degrees celsius and 5%carbon-dioxide. The next morning transfer the cells from the flask into individual 50 milliliter conical tubes. Wash the cells in up to 50 milliliters of HBSS per wash, and repeat this step up to three times to remove residual virus.
Incubate the cells for two hours in HSPC media supplemented with prostaglandin E2.Then wash the cells and resuspend the cells in 10 milliliters of HBSS supplemented with 2%autologous serum per tube. Then aspirate the cell suspensions using a 20 milliliter syringe equipped with a 16.5 gauge needle. Wash the inside of the tube with fresh HBSS plus 2%autologous serum, and aspirate into the syringe.
Carefully cap the syringe with the needle cap before placing the cell solution on ice until its infusion into an autologous host. To determine the gene-modification efficiency of the transduced cells, plate 1 x 10 to the 6th reserved mock or transduced cells per milliliter of HSPC medium onto non-tissue culture treated plates. On days two, five and twelve post-transduction, harvest and count the cells.
On days two and five, replate 33%of the cells in fresh HSPC medium at a 1 x 10 to the 6th per milliliter concentration. On days two, five and twelve, freeze 33%of the cells in DNA extraction buffer for quantitative real-time PCR, and analyze the final 33%of the cells by flow cytometry according to standard protocols to assess the phenotypic composition of the hematopoietic progeny. To quantify the transgene expression by flow cytometry, add three additional side scatter vs.
transgene plots;and gate the first plot on the hematopoietic stem cells, the second plot on the multipotent an-erythro myeloid progenitors, and the third plot on the lympho myeloid progenitors, all vs. the transgene. Then record 2000 to 3000 events and fine adjust the sort gates to fit the signal strength and target populations.
Using this protocol the number of nonhuman primate CD34 positive HSPC's that are enriched is typically proportional to the input total white blood cell count. As previous findings have demonstrated that the total CD34 positive HSPC product includes cells that are not true long term and grafting hematopoietic stem cells, these flow cytometry based and colony forming cell techniques can be used to reliably identify and differentiate subsets enriched for long term hematopoietic stem cells from committed progenitors. CD34 positive human stem cell enriched cells can be efficiently gene modified using lentiviral vectors.
Cells carrying a nonintegrated copy of the vector dilute out over the 12 day liquid culture assay period, whereas stably integrated vectors in the genome get passed on to all of the progeny. As confirmed by the expression of the vector in colony forming cell assays. This preclinical method can be used to isolate genetically modified in quality controlled nonhuman primate hematopoietic stem and progenitor cells for gene therapy.
This method can be easily adapted for other species, sources of hematopoietic stem and progenitor cells, gene therapy tools and diseases. This thoroughly vetted protocol shows great promise for the modeling of efficacious therapies for numerous human diseases. Keep in mind that working with nonhuman primate tissues requires enhanced security measures including personal protective equipment.
